Glucose dehydrogenase

ABSTRACT

Disclosed is a modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme, wherein one or more amino acid residues in a region of amino acid 349-377 of water-soluble PQQGDH derived from  Acinetobacter calcoaceticus  is replaced with other amino acid residues and has an inhibition constant (Ksi) of 200 mM or more. The modified water-soluble PQQGDH of the invention can be utilized for measuring glucose levels in the presence of high concentrations of glucose because of the low substrate inhibition by glucose.

TECHNICAL FIELD

The present invention relates to a modified glucose dehydrogenase (GDH),which is an enzyme having pyrroloquinoline quinone (PQQ) as a coenzyme,wherein certain amino acids are replaced with other amino acids. Themodified enzyme of the invention is useful in the quantification ofglucose for use in clinical diagnosis and food analysis.

BACKGROUND OF THE INVENTION

Glucose concentration is an important indicator in clinical diagnosis asan important marker for diabates. In addition, quantification of glucoseconcentration is an important indicator for monitoring the process offermentation production using bacteria. Conventionally, quantificationof glucose has been performed by enzymatic methods using glucose oxidase(GOD) or glucose-6-phosphate dehydrogenase (G6PDH). These days, the useof glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme(PQQGDH) is attracting attention in glucose quantification. PQQGDH hashighly oxidative activity towards glucose and it does not require oxygenas an electron acceptor since PQQGDH bears its coenzyme. Thus PQQGDH isa promising enzyme to be applied on glucose assays, for example, as arecognition devise of a glucose sensor.

PQQGDH is a glucose dehydrogenase having pyrroloquinoline quinone as acoenzyme, and catalyzes the reaction of oxidizing glucose to producegluconolactone. Two types of PQQGDHs are known: membrane-bound andwater-soluble. Membrane-bound PQQGDH is a single-peptide protein with anapproximate molecular weight of 87 kDa, and is found in a wide varietyof gram-negative bacteria. On the other hand, water-soluble PQQGDH hasbeen found in some strains of Acinetobacter calcoaceticus (Biosci.Biotech. Biochem. (1995), 59 (8), 1548-1555), and its structural genehas been cloned and its amino acid sequence determined (Mol. Gen. Genet.(1989), 217:430-436). Water-soluble PQQGDH derived from A. calcoaceticusis a water-soluble homodimer enzyme consisting of two 50 kDa subunits.It requires PQQ and Ca²⁺ for its activity and shows the enzyme activityof as high as 2200 U/mg-7400 U/mg. Isoelectric points are approximately9.2 and 10.2 for apoenzyme without bound PQQ and holoenzyme,respectively, indicating that the enzyme is a basic protein (K.Matsushita, et al. (1995) Biosci. Biotech. Biochem., 59, 1548-1555). Inaddition, the results of X-ray structural analysis of water-solublePQQGDH have been published and reveal the conformation of water-solublePQQGDH and estimated location of PQQ and Ca²⁺ (A. Oubrie, et al. (1999)J. Mol. Bio., 289, 319-333, A. Oubrie, et al. (1999) The EMBO Journal,18 (19), 5187-5194, and A. Oubrie, et al. (1999), PNAS 96 (21),11787-11791).

The wild type water-soluble PQQGDH shows a marked reduction in itsactivity by substrate inhibition under the glucose concentration of 100mM or more. For this reason, quantitative measurement of the substrateconcentration is difficult under high substrate concentration. Themechanism of substrate inhibition is not yet known.

Hence, the present invention is aimed at providing a modifiedwater-soluble PQQGDH which shows a little reduction of enzyme activitydue to substrate inhibition.

DISCLOSURE OF THE INVENTION

As a result of extensive research to modify the conventionalwater-soluble PQQGDH to develop a PQQGDH capable of quantifying glucoseeven at high glucose concentrations, the inventor successfully obtainedan enzyme with less substrate inhibition by introducing amino acidmutations at certain regions of water-soluble PQQGDH.

The present invention relates to a glucose dehydrogenase havingpyrroloquinoline quinone as a coenzyme, wherein one or more amino acidsin the amino acids residues 349-377 of water-soluble PQQGDH derived fromAcinetobacter calcoaceticus are replaced with other amino acids, andhaving an inhibition constant (Ksi) of 200 mM or more.

As used herein, the inhibition constant (Ksi) means the higher one ofthe substrate concentrations exhibiting half the maximum enzyme activityobserved. Under the conditions in which substrate inhibition in enzymeactivity can be observed, inhibition constant means an enzyme-specificvalue determined by the following formula:V=Vmax/[1+(Km/S)+(S/K′si)]wherein V is reaction rate; Vmax is the maximum reaction rate;

Km is Michaelis-Menten constant; S is substrate concentration; K′si is atheoretical value of the inhibition constant. The higher the K′si valueis, the higher the substrate concentration at which substrate inhibitionis observed will be, and substrate inhibition will be alleviated. Sinceit is difficult to measure K′si accurately in the presence ofimpurities, the observational Ksi value described above is used herein.

Although not intended to be bound by a specific theory, the amino acidregion 349-377 is predicted to be involved in the interaction thesubstrate glucose, because this region corresponds to the region formingthe 4D5A loop according to the topological prediction based upon PQQGDconformation revealed by A. Oubrie et al.

In this specification, the term “correspond” with reference to aminoacid residues or regions means that some amino acid residues or regionshave an equivalent function in two or more proteins which arestructurally similar but not identical. For example, a certain region inwater-soluble PQQGDH derived from organisms other than Acinetobactercalcoaceticus is said to “correspond to the region of amino acidresidues 349-377 of water-soluble PQQGDH derived from Acinetobactercalcoaceticus” when the amino acid sequence of such a region has a highsimilarity to the amino acid sequence in the 349-377 region ofwater-soluble PQQGDH derived from Acinetobacter calcoaceticus, and thesame function can be reasonably predicted based on the secondarystructure of the relevant regions in the proteins. Additionally, theamino acid residue 17 of the relevant region is said to “correspond tothe amino acid residue 365 of water-soluble PQQGDH derived fromAcinetobacter calcoaceticus”. The amino acid numbering in thisspecification starts from the initiator methionine as the +1 position.

Preferably, in the glucose dehydrogenase of the present invention, atleast one amino acid residues selected from the group consisting ofMet365, Thr366, Tyr367, Ile368, Cys369, or Ala374 in the amino acidsequence shown in SEQ ID NO: 1 is replaced with another amino acidresidue.

More preferably, the glucose dehydrogenase of the present invention hasat least one mutation selected from the group consisting of Met365Trp,Met365Phe, Thr366Asn, Thr366Ile, Thr366Asp, Thr366Lys, Tyr367Asp,Ile368Asn, Cys369Arg, and Ala374Pro in the amino acid sequence shown inSEQ ID NO: 1.

In another aspect, the modified PQQGDH of the present invention has amutation described above and also has another mutation in which Asp167of the amino acid sequence of SEQ ID NO: 1 is replaced with anotheramino acid residue, especially by glutamic acid. Involvement of Asp167in recognition and binding of a substrate by PQQGDH is described inJapanese Patent Public Disclosure No. 2001-346587. In general, however,no prediction can be made regarding the changes of substrate specificityand enzyme activity which may be caused by simultaneously altering theamino acid residues in the 4D5A loop domain and/or other domains.Therefore, it was a surprising discovery in the present invention thatboth improved specificity for glucose and high enzyme activity can beachieved at the same time by introducing double mutations.

In another aspect, the invention provides a glucose dehydrogenase havingpyrroloquinoline quinone as a coenzyme comprising one of the sequencesas follows: Cys Gly Glu Xaa Thr Tyr Ile (wherein Xaa is Met or Trp); GlyGlu Met Xaa Tyr Ile Cys (wherein Xaa is Asp, Lys, Ile or Asn); Glu MetThr Asp Ile Cys Trp; Met Thr Tyr Asp Cys Trp Pro; Thr Tyr Ile Arg TrpPro Thr; and Pro Thr Val Pro Pro Ser Ser.

The invention also provides a gene coding for PQQGDH of the invention, avector and a transformant comprising the gene of the invention, a methodfor preparing PQQGDH of the invention, as well as a glucose assay kitand a glucose sensor comprising PQQGDH of the invention.

Since the enzyme PQQGDH of the invention shows a low level of substrateinhibition by glucose, it is useful for measuring the level of glucoseeven at high glucose concentrations.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows construction of the pGB2 plasmid used in the invention.

FIG. 2 shows a method of constructing a mutant coding for the modifiedenzyme of the invention.

FIG. 3 shows the SV plot of the modified enzyme Tyr367Asp of theinvention.

FIG. 4 shows the SV plot of the modified enzyme Cys369Arg of theinvention.

DETAILED EXPLANATION OF THE INVENTION

Preparation Method of Modified PQQGDH

The sequence of the gene encoding wild type water-soluble PQQGDH proteinderived from Acinetobacter calcoaceticus are defined in SEQ ID NO:2.

Genes encoding modified PQQGDHs of the present invention can beconstructed by replacing the nucleotide sequences encoding certain aminoacids of the wild type water-soluble PQQGDH with the nucleotidesequences encoding the amino acids to be replaced. A wide range ofmethods for site-specific mutagenesis have been elaborated in the art.See, for example, Sambrook et al., “Molecular cloning; A LaboratoryManual”, second edition, 1989, Cold Spring Harbor Laboratory Press, NewYork.

The mutant gene obtained in this manner is inserted into an expressionvector (such as a plasmid) and transformed into an appropriate host(such as E. coli). A wide variety of host-vector systems have beendeveloped in the art to express exogenous proteins. For example,bacteria, yeast, and cultured cells can be used as hosts.

As long as its glucose dehydrogenase activity is retained, the modifiedPQQGDH of the invention can further contain deletion, substitution oraddition of other amino acid residues. A wide range of methods forsite-specific mutagenesis are available in the art. See, for example,Sambrook et al., “Molecular cloning; A Laboratory Manual”, secondedition, 1989, Cold Spring Harbor Laboratory Press, New York.

Moreover, those skilled in the art can determine a region in awater-soluble PQQGDH derived from other bacteria which corresponds tothe amino acid residues 349-377 of the water-soluble PQQGDH derived fromAcinetobacter calcoaceticus by comparing the array of the primarystructure of the proteins, or by comparing the secondary structurespredicted from the primary structures of the enzymes. Thus, additionalmodified PQQGDHs with reduced substrate inhibition can be obtained bysubstituting amino acid residues in this region with another amino acidresidues. Such modified PQQGDHs are also within the scope of the presentinvention.

After culturing the transformants expressing modified PQQGDH, obtainedas described above, the cells may be collected by centrifugation andthen crushed by French press, or the periplasmic enzymes may be releasedinto the culture by osmotic shock. After ultracentrifugation, solublefractions containing PQQGDH can be obtained. Alternatively, expressedPQQGDH can be secreted into the culture by using an appropriatehost-vector system.

The soluble fraction obtained as described above is then purified bycation exchange chromatography. Purification can be performed byfollowing the general instructions described in the textbooks known inthe art. A wide variety of columns for cation exchange chromatographyare available for protein purification, and any of them can be utilizedin this invention, including CM-5PW, CM-Toyopearl 650M, SP-5PW (TosoCo.), and S-Sepharose, Mono-S, S-Resource (Pharmacia Corp.). The columnis equilibrated with an appropriate buffer, the sample is applied to thecolumn, and then the unabsorbed materials are washed away. Suitablebuffers are, for example, phosphate buffer or MOPS buffer.

Then, substances absorbed in the column can be eluted by applying abuffer containing a higher salt concentration. The concentration of saltcan be changed gradually or linearly or combination thereof by usingbuffers containing different salt concentrations. Elution of the sampleis monitored by absorptiometer and the solution is fractionated intoappropriate volumes. Enzyme activity is measured for each fraction, andthe desired fractions are collected to obtain a purified preparation ofthe modified enzyme of the invention.

In addition, conventional methods known in the art, such as filtration,dialysis, gel filtration chromatography, or affinity chromatography, canbe used before or after cation exchange chromatography, if necessary.

Measurement of Enzyme Activity

The PQQGDH of the present invention with coenzyme PQQ catalyzesoxidation of glucose to produce gluconolactone. The enzyme activity canbe quantified by color-developing reaction of a redox dye to measure theamount of PQQ reduced with glucose oxidation by PQQGDH. Example ofcolor-developing reagents include PMS (Phenazine methosulfate), DCIP(2,6-dichlorophenolindophenol), potassium ferricyanide, and ferrocene.

Evaluation of Substrate Inhibition

The degree of substrate inhibition of the PQQGDH of the presentinvention can be evaluated by its inhibition constant (Ksi). Ksi isrepresented by the higher one of the substrate concentrations which showhalf of the highest level of the enzyme activity, when the enzymeactivity is measured using various glucose concentrations.

Evaluation of Substrate Specificity

The glucose specificity of the present invention can be evaluated bymeasuring relative enzyme activity with respect to the activity forglucose, as described above, by using a variety of sugars such as2-deoxy-D-glucose, mannose, allose, 3-o-methyl-D-glucose, galactose,xylose, lactose, and maltose as a substrate.

Glucose Assay Kit

The present invention also provides a glucose assay kit containing themodified PQQGDH of the invention. The glucose assay kit of the inventionmay contain a sufficient quantity of the modified PQQGDH to carry out atleast one assay. Besides modified PQQGDH, the kit may typically comprisebuffers required for assay, a mediator, a standard solution of glucoseto generate a calibration curve, and instructions for use. The modifiedPQQGDH can be supplied in a variety of forms, for example, asfreeze-dried reagent or appropriate stock solutions. Preferably, themodified PQQGDH of the present invention may be supplied in the form ofa holoenzyme, but can be supplied in the form of apoenzyme and convertedinto a holoenzyme before use.

Glucose Sensor

The present invention also provides a glucose sensor containing themodified PQQGDH of the invention. Carbon, gold, or platinum may be usedas an electrode, and the enzyme of the present invention is immobilizedon the electrode. Immobilization methods includes, for example, methodsusing cross-linking reagents, inclusion into a macromolecular matrix,coating with dialysis membrane, methods using photo-crosslinkingpolymer, electric conductive polymer, and redox polymer. The enzyme canalso be immobilized in a polymer or adsorbed on the electrode togetherwith an electron mediator, such as ferrocene or its derivative.Combinations of the above may also be used. Preferably, the modifiedPQQGDH of the present invention is immobilized on the electrode in theform of a holoenzyme, but can also be immobilized in the form ofapoenzyme and PQQ is supplied as another layer or in solution.Typically, the modified PQQGDH of the present invention is immobilizedon the electrode using glutaraldehyde, then free functional moieties ofglutaraldehyde are blocked by treatment with a reagent having aminegroups.

Measurement of glucose concentration is carried out as described below.Buffer, PQQ, CaCl₂, and a mediator are placed into aconstant-temperature cell and are kept at a constant temperature.Potassium ferricyanide and phenazine methosulfate may be used as amediator. An electrode in which the modified PQQGDH of the presentinvention is immobilized are used as a working electrode, together witha counter electrode (e.g., platinum) and a reference electrode (e.g.,Ag/AgCl electrode). A constant voltage is applied to the carbonelectrode. After the current reaches a constant value, aglucose-containing sample is added and the increase in the current ismeasured. The glucose concentration in the sample can be calculatedusing a calibration curve generated by standard concentration glucosesolutions.

All patents and references cited in this specification are incorporatedby reference. All the contents disclosed in the specifications anddrawings of Japanese Patent Application Nos. 2003-71744 and 2002-172955,on which the application claims priority, are incorporated herein byreference.

The working examples described below further illustrate the inventionwithout limiting the present invention.

EXAMPLE 1

Construction of Gene Encoding Modified PQQGDH Enzyme

Mutagenesis was carried out based on the structural gene of PQQGDHderived from Acinetobacter calcoaceticus (SEQ ID NO:2). pGB2 plasmid wasconstructed by inserting the structural gene of PQQGDH derived fromAcinetobacter calcoaceticus into the multi-cloning site of pTrc99Avector (Pharmacia) (FIG. 1). Wild type gene sequence encoding PQQGDH wasreplaced with altered gene sequence encoding modified PQQGDH by standardmethod as previously described. Site specific mutagenesis was performedusing the pGB2 plasmid as shown in FIG. 2. The sequences of syntheticoligonucleotides used for mutagenesis are shown below. In order toconstruct a mutant containing two mutations, two oligonucleotide primerswere used simultaneously for mutagenesis. Met365Trp 3′-GT TGA ACA CCTCTC CCA TGG ATG TAA AC-5′ Met365Phe 3′-GT TGA ACA CCT CTC CCT TGG ATGTAA AC-5′ Thr366Asn 3′-GGT TGA ACA CCT CTC TAC TTG ATG TAA ACG AC-5′Thr366Ile 3′-GGT TGA ACA CCT CTC TAC TAG ATG TAA ACG AC-5′ Thr366Asp3′-GA ACA CCT CTC TAC CTG ATG TAA ACG ACC G-5′ Thr366Lys 3′-GA ACA CCTCTC TAC TTT ATG TAA ACG ACC G-5′ Tyr367Asp 3′-GGT TGA ACA CCT CTC TACTGG CTG TAA ACG ACC-5′ Ile368Asn 3′-AC TGG ATG TTA ACG ACC GG-5′Cys369Arg 3′-GG ATG TAA ACG ACC GGT TGT C-5′ Ala374Pro 3′-C GGT TGT CAAGGT GGC AGT AGA CG-5′ Asp167Glu 3′-GGA AGT AGT TTT CTT GTA GTC AGT CC-5′

A template was prepared by inserting the KpnI-HindIII fragmentcontaining part of the gene encoding PQQGDH derived from Acinetobactercalcoaceticus into pKF18k vector plasmid (TaKaRa). A mixture of template(50 fmol), selection primer (5 pmol) supplied in Mutan-Express Km kit,phosphorylated target primer (50 pmol), and the annealing buffersupplied in the kit ( 1/10 of total volume (20 μl)) was prepared, andplasmid DNA was denatured to single-strand by heating at 100° C. for 3minutes. The selection primer was designed for the reversion ofdouble-amber mutation on the Kanamycin resistance gene of the pKF18kplasmid. Plasmid DNA was put on ice for 5 minutes for annealing of theprimers. A complementary strand was synthesized by adding the followingreagents: 3 μl of extension buffer supplied in the kit, 1 μl of T4 DNAligase, 1 μl of T4 DNA polymerase, and 5 μl of sterilized water. E. coliBMH71-18mutS, a DNA mismatch repair deficient strain, was transformedwith the synthesized DNA and cultured overnight with vigorous shaking toamplify the plasmid.

Then, each plasmid was extracted from bacteria and transformed into E.coli MV1184, and the plasmid was extracted from the colonies. Thesequence of the plasmid was determined to confirm successfulintroduction of the desired mutations. Kpn I-Hind III gene fragmentencoding wild type PQQGDH on pGB2 plasmid was replaced with the fragmentcontaining the mutation to construct a series of mutated PQQGDH genes.

EXAMPLE 2

Preparation of Modified Enzyme

A Gene encoding wild type or modified PQQGDH was inserted into themulti-cloning site of pTrc99A (Pharmacia), and the constructed plasmidwas transformed into E. coli DH5α. Transformants were cultured in 450 mlof L-broth using a Sakaguchi flask at 37° C. with vigorous shaking, andthen inoculated in 7 L of L-broth containing 1 mM CaCl₂ and 500 μM PQQ.After three hours of cultivation, IPTG was added to a finalconcentration of 0.3 mM, and cultivation was continued for another 1.5hours. The culture medium was centrifuged (5000 xg, 10 min, 4° C.), andthe pellet was washed with 0.85% NaCl twice. The cells were resuspendedin 10 mM phosphate buffer (pH7.0), crushed with French press (110 MPa),and centrifuged twice to remove the debris. The supernatant wasultracentrifuged (40,000 rpm, 90 min, 4° C.), and the supernatant wascollected as a water-soluble fraction. This fraction was dialyzedagainst A buffer (10 mM MOPS-NaCl buffer (pH 7.0)) at 4° C. overnight toobtain a crude preparation.

EXAMPLE 3

Measurement of Enzyme Activity

Enzyme activity was measured in MOPS-NaOH buffer (pH7.0) containing PMS(phenazine methosulfate)-DCIP (2, 6-dichlorophenolindophenol). Changesin absorbance of DCIP was recorded with a spectrophotometer at 600 nm,and the reduction rate of absorbance was defined as the reaction rate ofthe enzyme. In this measurement, enzyme activity which reduced 1 μmol ofDCIP in one minute was regarded as 1 unit. The molar absorptioncoefficient of DCIP at pH 7.0 was 16.3 M⁻¹.

EXAMPLE 4

Evaluation of Substrate Inhibition

Each of the crude enzyme preparation of wild type PQQGDH and modifiedPQQGDHs obtained in Example 3 was converted to a holoenzyme in thepresence of 1 μM PQQ and 1 mM CaCl₂ for 1 hour or more in the samemanner as described above. The solution was divided into aliquots of 187μl each, and mixed with 3 μl of activation reagents (6 mM DCIPA 48 μl,600 mM PMS 8 μl, 10 mM phosphate buffer pH 7.0 16 μl) and 10 μl ofD-glucose of various concentrations. Enzyme activity was measured atroom temperature as described above. Enzyme activity was plotted againstsubstrate concentration, and Km, Vmax, and Ksi values were determined.The results are shown in FIG. 1. The SV plot for Tyr367Asp and SV plotfor Cys369Arg are shown in Table 3 and Table 4, respectively. Theseresults clearly demonstrated that modified PQQGDH of the inventionshowed a higher Ksi value than wild type PQQGDH and significantreduction in substrate inhibition. TABLE 1 Km Vmax Ksi (mM) (U/mgprotein) (mM) Ksi/Km Wild type 23 154 196 8 Met365Phe 36 619 394 10Met365Trp 38 89 458 12 Thr366Asn 32 300 500 15 Thr366Ile 39 87 228 6Thr366Asp 35 196 556 16 Thr366Lys 23 300 202 9 Tyr367Asp 280 11 830 3Ile368Asn 61 60 535 9 Cys369Arg 65 6 1402 22 Ala374Pro n.d. 2 250 n.d.

EXAMPLE 5

Purification of Enzyme

The crude enzyme preparation obtained in Example 2 was adsorbed in acation exchange chromatography column filled with TSKgel CM-TOYOPEARL650M (Toso Co.). The column was washed with 750 ml of 10 mM phosphatebuffer pH 7.0 and the enzyme was eluted with 10 mM phosphate buffer pH7.0 containing from 0 M to 0.2 M NaCl. The flow rate was 5 mL/min.Fractions showing GDH activity were collected and dialyzed against 10 mMMOPS-NaOH buffer (pH 7.0) overnight. In this manner, modified PQQGDHprotein was purified which exhibited a single band underelectrophoresis. Enzyme activity and substrate inhibition of thepurified enzyme were measured in the presence of 0.6 mM PMS. The resultsare shown in Table 2. The modified enzymes, Thr366Asn and Thr366Asp, ofthe invention showed enzyme activity comparable to or higher than thewild type as well as a higher Ksi value. TABLE 2 Vmax kcat/Km Km (U/mgkcat (mM⁻¹ · Ksi (mM) protein) (sec⁻¹) sec⁻¹) (mM) Ksi/Km Wild type 278899 7451 276 250 9 Thr366Asn 14 10158 8505 608 522 37 Thr366Asp 28 51664283 153 332 12

EXAMPLE 6

Construction of Enzyme with Double Mutations

An enzyme with double mutations Asp167Glu/Thr366Asn was constructed andits characteristics were examined. Modified enzyme with Asp167Glumutation is known to have a higher substrate specificity to glucose.Substrate inhibition of the enzyme with double mutations was determinedin the same manner as Example 5 and the results were as follows: Ksi=600mM, Km=26 mM, and Ksi/Km=23. These values were equivalent to those ofthe modified enzyme of the invention as indicated in Example 5 and werehigher than those of the wild type enzyme.

Then, the substrate specificity of this enzyme was examined. Crudeenzyme preparations of the wild type and the modified PQQGDHs obtainedin Example 2 were converted into a holoenzyme in the presence of 1 μMPQQ, 1 mM CaCl₂ for one hour or more. This solution was divided intoaliquots or 187 μl, and mixed with 3 μl of activation reagent containingelectron acceptor (6 mM DCIP, 600 mM PMS, 10 mM phosphate buffer pH7.0), and substrate were added (final concentration: 0.06 mM DCIP, 0.6mM PMS). Ten μl of glucose, lactose or maltose was added as a substrateto a final concentration of 100 mM and the samples were incubated for 30minutes at room temperature. The enzyme activity was measured in thesame manner as Example 3. The values were expressed as a relativeactivity to the activity for glucose (100). The results are shown inTable 3. Modified enzyme with double mutations, Thr366Asn and Asp167Glu,exhibited higher substrate specificity to glucose than either wild typeor modified enzyme with a single mutation Asp167Glu. In addition,modified enzyme with single mutation Thr366Asn has an equivalent levelof substrate specificity to that of the wild type enzyme (data notshown). TABLE 3 Glucose Lactose Maltose Wild type 100% 54% 58% Asp167Glu100% 32% 10% Asp167Glu/Thr366Asn 100% 20%

In addition, the enzyme activity of this double mutant was measured inthe presence of 0.06 mM DCIP as electron acceptor and 10 mM glucose assubstrate in the same manner as in Example 4. The modified enzyme withdouble mutations Thr366Asn and Asp167Glu exhibited higher enzymeactivity than the wild type enzyme. TABLE 4 Wild type 100% Thr366Asn126% Asp167Glu  54% Asp167Glu/Thr366Asn 291%

EXAMPLE 7

Preparation of Enzyme Sensor and its Evaluation

Twenty mg of carbon paste was added to 5 units of the modified enzymeand freeze-dried. The mixture was applied on the surface of a carbonpaste electrode filled with approximately 40 mg of carbon paste, and theelectrode was polished on a filter paper. This electrode was treatedwith MOPS buffer (pH 7.0) containing 1% glutaraldehyde for 30 minutes atroom temperature and then treated with MOPS buffer (pH 7.0) containing20 mM lysine for 20 minutes at room temperature to block unreactedglutaraldehyde. The electrode was equilibrated in 10 mM MOPS buffer (pH7.0) for one hour or more at room temperature, then stored at 4° C.

The glucose concentration was measured using the glucose sensor thusprepared. Glucose concentration was quantified in the range from 5 mM to50 mM by using the glucose sensor prepared with the modified PQQGDH ofthe invention.

Industrial Applicability

The modified water-soluble PQQGDH of the present invention can beutilized for glucose measurement in the presence of high concentrationsof glucose because of its low substrate inhibition.

1. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme, wherein one or more amino acid residues in a region of 349-377 amino acid of water-soluble PQQGDH derived from Acinetobacter calcoaceticus is replaced with other amino acid residues and has an inhibition constant (Ksi) of 200 mM or more.
 2. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein Met365 of the amino acid sequence defined in SEQ ID NO: 1 is replaced with another amino acid, and has a Ksi value of 200 mM or more.
 3. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein Met365 of the amino acid sequence defined in SEQ ID NO: 1 is replaced with tryptophan or phenylalanine.
 4. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein Thr366 of the amino acid sequence defined in SEQ ID NO: 1 is replaced with another amino acid, and has a Ksi value 200 mM or more.
 5. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein Thr366 of the amino acid sequence defined in SEQ ID NO: 1 is replaced with aspartic acid, lysine, isoleucine, or asparagines.
 6. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein Tyr367 of the amino acid sequence defined in SEQ ID NO: 1 is replaced with another amino acid, and has a Ksi value of 200 mM or more.
 7. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein Tyr367 of the amino acid sequence defined in SEQ ID NO: 1 is replaced with aspartic acid.
 8. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein Ile368 of the amino acid sequence defined in SEQ ID NO: 1 is replaced with another amino acid, and has a Ksi value of 200 mM or more.
 9. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein Ile368 of the amino acid sequence defined in SEQ ID NO: 1 is replaced with asparagine.
 10. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein Cys369 of the amino acid sequence defined in SEQ ID NO: 1 is replaced with another amino acid and has a Ksi value of 200 mM or more.
 11. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein Cys369 of the amino acid sequence defined in SEQ ID NO: 1 is replaced with arginine.
 12. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein Ala374 of the amino acid sequence defined in SEQ ID NO: 1 is replaced with another amino acid, and has a Ksi value of 200 mM or more.
 13. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein Ala374 of the amino acid sequence defined in SEQ ID NO: 1 is replaced with proline.
 14. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein one of the amino acids in 349-377 region of the amino acid sequence defined in SEQ ID NO: 1 and Asp167 are replaced with other amino acids.
 15. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein an amino acid residue selected from the group consisting of Met365, Thr366, Tyr367, Ile368, Cys369, and Ala374 of the amino acid sequence defined in SEQ ID NO: 1 is replaced with another amino acid and wherein Asp167 is replaced with another amino acid.
 16. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein an amino acid residue selected from the group consisting of Met365, Thr366, Tyr367, Ile368, Cys369, and Ala374 of the amino acid sequence defined in SEQ ID NO: 1 is replaced with another amino acid and wherein Asp167 is replaced with glutamic acid.
 17. A modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme wherein Thr366 of the amino acid sequence defined in SEQ ID NO: 1 is replaced with aspartic acid, lysine, isoleucine, or asparagine, and wherein Asp167 is replaced with glutamic acid.
 18. A glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme comprising the following amino acid sequence: Cys Gly Glu Xaa Thr Tyr Ile (SEQ ID NO: 3)

wherein Xaa is Met or Trp.
 19. A glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme comprising the following amino acid sequence: Gly Glu Met Xaa Tyr Ile Cys (SEQ ID NO: 4)

wherein Xaa is Asp, Lys, Ile or Asn.
 20. A glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme comprising the following amino acid sequence: Glu Met Thr Asp Ile Cys Trp. (SEQ ID NO: 5)


21. A glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme comprising the following amino acid sequence: Met Thr Tyr Asp Cys Trp Pro. (SEQ ID NO: 6)


22. A glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme comprising the following amino acid sequence: Thr Tyr Ile Arg Trp Pro Thr. (SEQ ID NO: 7)


23. A glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme comprising the following amino acid sequence: Pro Thr Val Pro Pro Ser Ser. (SEQ ID NO: 8)


24. A gene encoding a modified glucose dehydrogenase as claimed in claim
 1. 25. A vector comprising the gene as claimed in claim
 24. 26. A transformant comprising the gene as claimed in claim
 24. 27. A transformant as claimed in claim 26 wherein the gene as claimed in claim 24 is integrated in its chromosome.
 28. A method for preparing a water-soluble PQQGDH, comprising culturing the transformant as claimed in claim 27 and preparing water-soluble fraction from the cells of the transformant.
 29. A glucose assay kit comprising the modified glucose dehydrogenase as claimed in claim
 1. 30. A glucose sensor comprising the modified glucose dehydrogenase as claimed in claim
 1. 